Western Blot

While monoclonal antibodies have traditionally been used for protein detection via western blotting, aptamers are also well-suited for western blotting and other methods requiring selective affinity reagents.

Base Pair recently demonstrated a colorimetric aptamer Western Blot using an aptamer selective for penicillin-binding protein 2a, PBP2a. For proof of principle, Base Pair scientists used a NuPage 4-12% Bis-Tris Gel with NuPage buffers. Recombinant PBP2a was loaded at concentrations ranging from 6.2 to 100 pmol. Following electrophoresis, the gel was transferred to an iBlot nitrocellulose membrane using the iBlot protocol. The membrane was blocked with 1% non-fat dry milk in PBST.

Following aptamer folding, the membrane was incubated with fluorescein-labeled PBP2a/MRSA aptamer ATW0032 at a 200nM concentration for 60 minutes. After washing, the membrane was incubated with a 1:2,000 dilution of anti-FITC-HRP antibody for 30 minutes. TMB colorimetric substrate was added after washing and incubated until desired band development was achieved. The membrane was rinsed, dried, and photographed.

While a traditional HRP/TMB colorimetric enzyme/substrate system was used for this western blot, a chemiluminescent substrate and CCD imaging system are recommended for enhanced imaging and detection.

For more information on the Base Pair aptamer used in this western blot, visit the PBP2a/MRSA Aptamer ATW0032 product page.

For questions about this experiment or other aptamer-based applications, please contact a Base Pair aptamer specialist at support@basepairbio.com.